Turnover of the human proteome: determination of protein intracellular stability by dynamic SILAC.
The proteome of any system is a dynamic entity, such that the intracellular concentration of a protein is dictated by the relative rates of synthesis and degradation. In this work, we have analyzed time-dependent changes in the incorporation of a stable amino acid resolved precursor, a protocol we refer to as "dynamic SILAC", using 1-D gel separation followed by in-gel digestion and LC-MS/MS analyses to profile the intracellular stability of almost 600 proteins from human A549 adenocarcinoma cells, requiring multiple measures of the extent of labeling with stable isotope labeled amino acids in a classic label-chase experiment. As turnover rates are acquired, a profile can be built up that allows exploration of the 'dynamic proteome' and of putative features that predispose a protein to a high or a low rate of turnover. Moreover, measurement of the turnover rate of individual components of supramolecular complexes provides a unique insight in processes of protein complex assembly and turnover.
Doherty
Mary K
MK
School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool L69 3BX, UK.
Hammond
Dean E
DE
Clague
Michael J
MJ
Gaskell
Simon J
SJ
Beynon
Robert J
RJ
D045744:Cell Line, Tumor; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry*; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012270:Ribosomes / Q000737:chemistry; D013269:Stochastic Processes; D013997:Time Factors
D011506:Proteins; D020543:Proteome